Compositions and methods for detecting phosphomonoester

ABSTRACT

The invention provides a method of modifying a phosphomonoester moiety of a target compound. The method can include the steps of (a) providing a target compound having an electrophilic moiety and a phosphomonoester moiety; (b) contacting the target compound with a first carbodiimide compound under conditions for preferential addition of the first carbodiimide compound to the electrophilic moiety over the phosphomonoester moiety, thereby forming an electrophile-protected target compound; and (c) contacting the electrophile-protected target compound with a second carbodiimide compound and a nucleophilic compound under conditions for addition of the nucleophilic compound to the phosphomonoester.

This invention was made with government support under grant number 1 R43GM071272-01A1 awarded by the National Institute of Health. The UnitedStates Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates generally to analysis of phosphorylatedcompounds, and more specifically to detecting phosphorylation ofbiological molecules.

The cells of all organisms are capable of evaluating changes in theirenvironment and appropriately altering function for an appropriateresponse. The responses are mediated by signal transduction systems thatconvert external stimuli into internal biochemical signals. These signaltransduction systems share several characteristics across a wide varietyof organisms whether single cell organisms such as bacteria ormulticellular organisms such as animals and plants. Typically, aspecific cellular receptor changes from one state to another due to aparticular environmental stimulus and this change in state initiates aseries of biochemical reactions that lead to one or more functionalresponses of the cell to the stimulus. For example, the presence of aparticular nutrient, toxin or hormone can be detected by a cell leadingto a change in cellular function allowing uptake and metabolism of thenutrient, sequestration and breakdown of the toxin or changes in growthand development, respectively.

One of the most widely used biochemical reactions of the signaltransduction pathways of a wide variety of organisms is the transfer ofa phosphate residue onto a biological target molecule in such a way thatthe function of the target molecule is changed. The addition ofphosphates is mediated by kinases. Typically, a series of proteinkinases act to sequentially activate each other by phosphorylation untilultimately the pathway reaches an enzyme that, when phosphorylated,changes its function to adapt the cell to the initial stimulus thatactivated the signal transduction pathway.

Signal transduction pathways that utilize phosphorylation play criticalroles in a multitude of cellular functions including cell homeostasis,differentiation, development and growth. Improper function or responseof signal transduction pathways have been found to play important rolesin a variety of diseases and conditions including, for example, cancer,allergic responses, autoimmune diseases and degenerative diseases. Inaddition, the efficacy of many therapeutic drugs is due to their abilityto alter particular signal transduction pathways.

The ability to monitor the states of phosphorylation for specificbiochemical molecules can provide an understanding of many fundamentalbiological processes, for example, in a research setting. This can alsobe beneficial in a clinical setting for the diagnosis of variousdiseases or conditions, or for the prognosis of individuals beingtreated for a particular disease or condition. Furthermore, the abilityto monitor phosphorylation can be valuable for the design oridentification of therapeutic drugs that target specific kinases orsignal transduction pathways.

Currently, several technologies are available for analyzing anddetecting phosphorylation of biological molecules. One of the mostwidely used methods is to treat a test sample with ATP containingradioactive γ-phosphate followed by evaluating incorporation ofradioactivity into a target component of interest. In many cases, theuse of radioactivity is undesirable due to safety concerns, the cost ofpreparing and handling radioactive material and the environmentalimpacts of radioactivity disposal. Radioactivity can be avoided by usinga phospho-specific antibody to detect a phosphorylated target component.However, the ability of these antibodies to detect a phosphate residueis dependent upon the chemical composition of the component to which thephosphate residue is attached. In many cases a new antibody must begenerated for each different target to be detected. This can lead todifficulties when dealing with biological targets for which the exactcomposition is unknown. Even when targets of known composition are used,the need to generate and evaluate different antibodies for each targetcan be both time consuming and expensive.

Thus, there exists a need for a method of detecting phosphorylation ofbiological targets that is safe, effective and widely applicable totargets having any of a variety of different chemical compositions. Thepresent invention satisfies this need and provides other advantages aswell.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of modifying a phosphomonoester moietyof a target compound. The method can include the steps of (a) providinga target compound having an electrophilic moiety and a phosphomonoestermoiety; (b) contacting the target compound with a first carbodiimidecompound under conditions for preferential addition of the firstcarbodiimide compound to the electrophilic moiety over thephosphomonoester moiety, thereby forming an electrophile-protectedtarget compound; and (c) contacting the electrophile-protected targetcompound with a second carbodiimide compound and a nucleophilic compoundunder conditions for addition of the nucleophilic compound to thephosphomonoester.

The invention further provides a method of modifying a phosphomonoestermoiety of a target compound. The method can include the steps of (a)providing a target compound having a carboxylic acid moiety and aphosphomonoester moiety; (b) contacting the target compound with a firstcarbodiimide compound under conditions for preferential addition of thefirst carbodiimide compound to the carboxylic acid moiety over thephosphomonoester moiety, thereby forming a carboxyl-protected targetcompound; and (c) contacting the carboxyl-protected target compound witha second carbodiimide compound and an amine under conditions foraddition of the amine to the phosphomonoester.

The invention further provides a method of detecting a phosphomonoestermoiety of a target compound. The method can include the steps of (a)providing a target compound having a carboxylic acid moiety and aphosphomonoester moiety; (b) contacting the target compound with a firstcarbodiimide compound under conditions for preferential addition of thefirst carbodiimide compound to the carboxylic acid moiety over thephosphomonoester moiety, thereby forming a carboxyl-protected targetcompound; (c) contacting the carboxyl-protected target compound with asecond carbodiimide compound and an amine under conditions for additionof the amine to the phosphomonoester moiety, thereby forming anucleophile-modified phosphate moiety; and (d) detecting thenucleophile-modified phosphate moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reactions for derivatization of a carbonyl moiety with anO-acylurea or N-acylurea moiety (reaction A) and forcarbodiimide-activated addition of an amino-containing dye tophosphomonoester moiety (reaction B).

FIG. 2 shows a schematic representation of a kinase assay.

FIG. 3 shows a bar graph of signal intensities for different targetcompounds detected by EDC activated addition of an amino-containing dyefollowing blocking of carbonyl moieties by EDC pre-treatment.

FIG. 4 shows results for phosphorylation of target proteins by p60c-srckinase and PKA kinase detected by chemical labeling (Panel A) orantibody-based labeling (Panel B); correlation of the two labelingapproaches is also shown (Panel C).

FIG. 5 shows a bar graph of kinase activity data for p60c-src and PKAkinases in a multiplex assay having 194 different protein-DNA targets,including substrates for various kinases, and in the presence or absenceof inhibitors (Panel A) and an expanded view of a region of the bargraph showing kinase activity in the presence and absence of kinaseinhibitors (Panel B).

FIG. 6 shows a table of results comparing cross reactivity andinhibition efficiency for assay of p60c-src kinase and PKA kinase in amultiplex assay having a pool of 194 different protein-DNA targets,including substrates for various kinases.

FIG. 7 shows signal measured for each of 96 different protein-DNAtargets when treated with p60c-src kinase (Panel A) and PKA kinase(Panel B) in multiplex assays.

FIG. 8 shows bar graphs of results for kinase assays of 96 differentprotein-DNA targets when treated with p60c-src kinase and detected usingantibody labeling (Panel A) or chemical labeling (Panel B).

FIG. 9 shows star plots of the phosphorylation of 96 differentprotein-DNA kinase targets by the kinases p60c-src, PKA, Abl and PKC.

FIG. 10 shows star plots of the performance of 96 different protein-DNAtargets in the presence of selective inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for specifically modifying aphosphomonoester moiety of a target compound using carbodiimidechemistry. The method can include a step of protecting otherelectrophilic moieties in the target compound and is, therefore, wellsuited to modification of target compounds having other electrophilicmoieties and/or target compounds in samples having such electrophilicmoieties. An advantage of the methods is that both steps utilizecarbodiimide chemistry and can be carried out in a one pot reaction.Thus, the target compound need not be separated from reagents used forthe electrophile-protection step prior to modifying the phosphomonoestermoiety.

The invention further provides the ability to modify a phosphomonoestermoiety for detection of the phosphorylation state of a target compound.In particular embodiments, the methods can be used to determine activityof a kinase or phosphatase. The activity can be determined from thephosphorylation state of a target compound that is treated with a kinaseor phosphatase.

An advantage of the methods is that modification or detection can beapplied to target compounds having any of a variety of differentchemical compositions. Thus, in embodiments where the target compoundsare proteins, the methods can be used to determine the phosphorylationstate of protein targets in a manner that is independent of differencesin the amino acid sequences of the different proteins.

The invention is well suited to multiplex formats. Thus, a method of theinvention can be used to modify a plurality of target compounds havingphosphomonoester moieties. Furthermore, a plurality of target compoundsmodified in a method of the invention can be detected in a multiplexformat. For example, a mixture of target compounds can be treated with amixture of kinases, phosphatases or both, resulting in a mixture oftarget compounds having phosphomonoester moieties and thephosphomonoester moieties can be modified to produce a mixture ofphosphate-modified target compounds that can be detected.

The invention can be used for any of a variety of purposes, such as thediagnosis of disease; determination of the response of cells to anexternal agent, such as a drug; staging of a disease, such as neoplasia;identifying cell differentiation or maturation; identifying new targetcompounds, identifying kinases or phosphatases present in a sample;screening for drugs that target a kinase or phosphatase, determiningpotential side effects of a drug; determining selectivity of a drug orthe like.

DEFINITIONS

As used herein, the term “compound” is intended to mean a combination oftwo or more atoms held together by chemical bonds. A compound caninclude several moieties having the same or different properties such asstructure, reactivity with a particular reagent or ability to generate adetectable signal. An exemplary compound useful in the invention is atarget compound that is modified, detected or manipulated or for whichmodification, detection or manipulation is desired. A target compoundcan be made or used as a discrete compound or as a part of a largermolecule or complex. Exemplary target compounds include, but are notlimited to, a molecule, polymer, protein or nucleic acid and can bebioactive, naturally occurring, non-naturally occurring or synthetic.

As used herein, the term “protein” is intended to mean a chain of aminoacids connected by peptide bonds. The term is intended to include chainshaving any possible number of amino acids, unless explicitly indicatedotherwise. Accordingly a protein can include a single linear chainhaving at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100,1,000, 10,000, 100,000 or more amino acids. If desired, a protein usefulin the invention can have a maximum length including, for example, atmost about 100,000, 10,000, 1,000, 100, 10, 5 or fewer in a linearchain. A protein can include one or more of the 20 amino acids used by ahuman cell to translate RNA into protein. Furthermore, a protein caninclude other amino acids such as non-naturally occurring amino acids. A“species” of proteins or protein moieties is understood to be a groupthat all include the same sequence of amino acids.

As used herein, the term “nucleic acid” is intended to mean a polymermolecule composed of subunits having purine or pyrimidine bases. Anucleic acid useful in the present invention will generally containphosphodiester bonds, and can include, for example, DNA or RNA. Ifdesired to suit a particular application, DNA or RNA analogs havingalternate backbones can be used, including, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10): 1925 (1993) and referencestherein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur.J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), or peptide nucleic acid linkages (see Egholm,J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Otherpolynucleotide analogs include those with positive backbones (Denpcy etal., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarscan also be used in the invention (see Jenkins et al., Chem. Soc. Rev.(1995) pp 169-176). Several other nucleic acid analogs are described inRawls, C & E News Jun. 2, 1997 page 35. Each of the above-citedreferences is hereby incorporated by reference

A nucleic acid can be single stranded, double stranded or containportions of both double stranded and single stranded sequence. Apolynucleotide can be DNA, RNA or a hybrid containing any combination ofdeoxyribo- and ribo-nucleotides or any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthanine,hypoxanthanine, isocytosine, isoguanine, or the like. A “species” ofnucleic acids or nucleic acid moieties is understood to be a group thatall include the same nucleic acid sequence.

As used herein, the term “carboxylic acid moiety” is intended to mean apart of a compound having a carbon atom singly bonded to another atom ofthe compound, doubly bonded to a first oxygen atom and singly bonded toa second oxygen atom, wherein the second oxygen atom is negativelycharged (O—) or bonded to a hydrogen as a hydroxyl moiety (OH).Exemplary carboxylic acid moieties that can be found in a proteininclude the alpha carboxylic acid moiety of the C-terminal amino acid ofthe protein, the beta carboxylic acid moiety of aspartate or the gammacarboxylic acid moiety of glutamate.

As used herein, the term “electrophile-protected,” when used inreference to a compound, is intended to mean a compound for which anelectrophilic moiety has been removed or modified to reduce or preventreactivity toward a nucleophilic reagent. An exemplaryelectrophile-protected moiety is a carboxyl-protected moiety.

As used herein, the term “carboxyl-protected,” when used in reference toa compound, is intended to mean a compound for which a carboxylic acidmoiety has been removed or modified to reduce or prevent reactivitytoward a carboxylic acid-reactive reagent. In particular embodiments, acarboxyl-protected moiety includes a carbon atom singly bonded toanother atom of the compound, doubly bonded to a first oxygen atom andsingly bonded to a second oxygen atom, wherein the second oxygen atom isfurther bonded to an atom other than hydrogen such as an atom of anorganic moiety. Exemplary carboxyl-protected moieties include, but arenot limited to, an ester moiety, amide moiety, N-acylurea moiety, orO-acylurea moiety.

As used herein, the term “N-acylurea moiety” is intended to mean a partof a compound including the structure

R₁ and R₂ can be the same or different and can be selected from thegroup consisting of hydrogen, ethyl, 3-dimethylamino propyl, cyclohexyl,2-morpholinoethyl, isopropyl, phenyl or other organic moiety such as anoptionally substituted alkyl or aryl. Furthermore, 2 of the R groups canbe joined together to form a ring structure.

As used herein, the term “O-acylurea moiety” is intended to mean a partof a compound including the structure

R₁ and R₂ can be the same or different and can be selected from thegroup consisting of hydrogen, ethyl, 3-dimethylamino propyl, cyclohexyl,2-morpholinoethyl, isopropyl, phenyl or other organic moiety such as anoptionally substituted alkyl or aryl. Furthermore, 2 of the R groups canbe joined together to form a ring structure.

As used herein, the term “phosphomonoester moiety” is intended to mean apart of a compound having a phosphorus atom bound to another atom of thecompound and bound to 3 different oxygen atoms. Typically, a firstoxygen atom is double bonded to the phosphorus, the second and thirdoxygens are singly bonded to the phosphorus, and the second and thirdoxygen atoms are either negatively charged (O—) or bonded to a hydrogenas a hydroxyl moiety (OH). A phosphomonoester having one or morenegatively charged oxygens can have any of a variety of the same ordifferent counter ions (for example, sodium, potassium or the like).Various ionic forms of phosphomonoester can occur including, forexample, the free acid, mono-anion, or di-anion. A phosphomonoestermoiety can be in the form of a monophosphate or a portion of aphosphoric anhydride such as a diphosphate, triphosphate etc.

As used herein, the term “phosphoric amide” is intended to mean a partof a compound having a phosphorus atom bound to another atom of thecompound, to a nitrogen atom of an organic moiety and to 2 differentoxygen atoms, wherein a first oxygen atom is double bonded to thephosphorus and a second oxygen is singly bonded to the phosphorus, andthe second oxygen atom is either negatively charged (O—) or bonded to ahydrogen as a hydroxyl moiety (OH).

As used herein, the term “carbodiimide compound” is intended to mean amolecule including a —N═C═N— moiety. A carbodiimide useful in theinvention can have a structure

R₁—N═C═N—R₂

R₁ and R₂ can be the same or different and can be selected from thegroup consisting of hydrogen, ethyl, 3-dimethylamino propyl, cyclohexyl,2-morpholinoethyl, isopropyl, phenyl or other organic moiety such as anoptionally substituted alkyl or aryl. Furthermore, the R groups can bejoined together to form a ring structure. Exemplary carbodiimidecompounds include, but are not limited to,1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC);1-cyclohexyl-3(2-morpholinoethyl) carbodiimide-metho-p-toluenesulfonate; (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline);dicyclohexyl carbodiimide; diisopropyl carbodiimide or others known inthe art such as those described in Dolinnaya et al., Nucleic AcidResearch 19:3073-3080 (1991), which is incorporated herein by reference.In general a carbodiimide is used in the form of an acid addition salt,e.g., hydrochloride, thereof.

As used herein, the term “amine” is intended to mean a compoundcontaining nitrogen. The compound can be an organic compound havingstructure R_(3-x) NH_(x), where R is an organic moiety and 0<x<3.Exemplary forms of these organic compounds include a primary amine(x=2), secondary amine (x=1), or tertiary amine (x=0). An amine can beneutral or positively charged.

As used herein, the term “label moiety” is intended to mean one or moreatoms that can be specifically detected to indicate the presence of asubstance to which the one or more atoms are attached. A label moietycan be a primary label that is directly detectable or secondary labelthat can be indirectly detected, for example, via interaction with aprimary label. Exemplary primary labels include, without limitation, anisotopic label such as a naturally non-abundant heavy isotope orradioactive isotope, examples of which include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ³²P, ³⁵S or ³H; optically detectable moieties such as achromophore, luminophore, fluorophore, quantum dot or nanoparticle;electromagnetic spin label; calorimetric agent; magnetic substance;electron-rich material such as a metal; electrochemiluminescent labelsuch as Ru(bpy)₃ ²⁺; moiety that can be detected based on a nuclearmagnetic, paramagnetic, electrical, charge to mass, or thermalcharacteristic; or light scattering or plasmon resonant materials suchas gold or silver particles. Fluorophores that are useful in theinvention include, for example, fluorescein, fluorescein isothiocyanate,carboxyfluorescein (FAM), dichlorotriazinylamine fluorescein, rhodamine,tetramethylrhodamine, umbelliferone, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, LuciferYellow, Cascade Blue™, Texas Red, alexa dyes, dansyl chloride,phycoerythrin, green fluorescent protein and its wavelength shiftedvariants, bodipy, fluorescent lanthanide complexes, including those ofEuropium and Terbium, and other fluorophores known in the art such asthose described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.)6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principlesof Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), orWO 98/59066, each of which is hereby incorporated by reference.

Exemplary secondary labels that can be used in the invention include,without limitation, a binding moiety such as a receptor, ligand or othermember of a pair of molecules having binding specificity for each other.Exemplary binding moieties having specificity for each other include,without limitation, streptavidin & biotin, avidin & biotin or an antigen& antibody such as rabbit IgG & anti-rabbit IgG. Specific affinitybetween two binding partners is understood to mean preferential bindingof one partner to another compared to binding of the partner to othercomponents or contaminants in the system. Depending upon the particularbinding conditions used, the dissociation constants of the pair can be,for example, less than about 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ 10⁻¹⁰,10⁻¹¹, or 10⁻¹² M⁻¹. Secondary labels also include enzymes or theirsubstrates, wherein the combination produces a detectable product,examples of which include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase each of which producecolorimetric products using color reagents that are commerciallyavailable, for example, from Sigma-Aldrich (St. Louis, Mo.) orInvitrogen (Carlsbad, Calif.).

The terms “receptor” and “ligand” are used herein for semantic clarityin identifying binding partners and are intended to be interchangeable,unless explicitly indicated to the contrary. Accordingly, the term“receptor” is intended to mean a molecule that is capable of selectivelybinding a ligand and the term “ligand” is intended to mean a moleculethat is capable of selectively binding a receptor. The terms areintended to encompass receptors or ligands that have other functions aswell. However, the terms are not intended to be limited by any otherfunction unless indicated otherwise. For example, a receptor can be anaturally occurring polypeptide having signal transducing activity or afunctional fragment thereof that exhibits selective binding to a ligandwhether or not the functional fragment has signal transducing activity.

As used herein, the term “array” refers to a population of differentprobe molecules that are attached to one or more solid-phase substratessuch that the different probe molecules can be differentiated from eachother according to their relative location. An array can includedifferent probe molecules that are each located at a differentaddressable location on a solid-phase substrate. Alternatively, an arraycan include separate solid-phase substrates each bearing a differentprobe molecule, wherein the different probe molecules can be identifiedaccording to the locations of the solid-phase substrates on a surface towhich the solid-phase substrates are attached or according to thelocations of the solid-phase substrates in a liquid such as a fluidstream.

Description of Particular Embodiments

The invention provides a method of modifying a phosphomonoester moietyof a target compound. The method can include the steps of (a) providinga target compound having an electrophilic, moiety and a phosphomonoestermoiety; (b) contacting the target compound with a first carbodiimidecompound under conditions for preferential addition of the firstcarbodiimide compound to the electrophilic moiety over thephosphomonoester moiety, thereby forming an electrophile-protectedtarget compound; and (c) contacting the electrophile-protected targetcompound with a second carbodiimide compound and a nucleophilic compoundunder conditions for addition of the nucleophilic compound to thephosphomonoester.

A target compound can have any of a variety of electrophilic moietiesincluding, for example, an amine-reactive moiety such as a carboxylicacid moiety. Other amine-reactive moieties that may be present in atarget compound include, for example, a carboxylic acid derivative suchas an ester, amide, anhydride or acyl halide, or an aldehyde.

An electrophile-protected target compound can be produced by reacting anelectrophilic moiety of the target compound with a nucleophilic compoundhaving an amine, hydrazine, sulfhydril, hydroxyl or other phosphatereactive moiety.

Accordingly, in a particular embodiment, the invention provides a methodof modifying a phosphomonoester moiety of a target compound. The methodcan include the steps of (a) providing a target compound having acarboxylic acid moiety and a phosphomonoester moiety; (b) contacting thetarget compound with a first carbodiimide compound under conditions forpreferential addition of the first carbodiimide compound to thecarboxylic acid moiety over the phosphomonoester moiety, thereby forminga carboxyl-protected target compound; and (c) contacting thecarboxyl-protected target compound with a second carbodiimide compoundand an amine under conditions for addition of the amine to thephosphomonoester.

The invention can be used to modify a phosphomonoester moiety of any ofa variety of target compounds including, for example, a naturallyoccurring compound such as a nucleotide, polynucleotide, phosphorylatedL-amino acid, phosphorylated protein, phosphorylated sugar,phosphorylated lipid, phosphorylated cofactor or phosphorylatedmetabolite. The invention can also be used to modify a phosphomonoestermoiety of a synthetic target compound whether it is non-naturallyoccurring or a synthetic version of a naturally occurring compound.Examples include non-natural amino acids, proteins including non-naturalamino acids, small molecule drug candidates, modified polynucleotides,non-natural carbohydrates or non-natural lipids. Exemplary non-naturalamino acids that can have a phosphomonoester moiety include, but are notlimited to, D-isomers of naturally occurring amino acids, such asD-serine, D-threonine, D-glutamic acid, D-aspartic acid or D-tyrosine;L- or D-isomers of homoserine, hydroxylated tryptophan, 3-nitro-tyrosineor other amino acids having a hydroxyl or carboxylic acid moiety.Proteins and amino acids useful in the invention can be obtained from acommercial supplier such as EMD biosciences (Darmstadt, Germany, see theNovabiochem catalog) Advanced Chemtech (Louisville, Ky.), PeptidesInternational (Louisville, Ky.), Chem-Impex (Wood Dale, Ill.), TCIAmerica (Portland Oreg.), or Matrix Scientific (Columbia, S.C.).

The invention is particularly useful for a target compound that is aprotein (i.e. a protein target). A protein target can have one or morephosphomonoester moieties that are modified in accordance with theinvention. Exemplary phosphomonoester moieties that are commonly foundin proteins and that can be modified as set forth herein include, forexample, the L-isomers of phosphoserine, phosphothreonine,phosphotyrosine, phosphoglutamate, phosphoaspartate or phosphohistidine.In some embodiments, one or more phosphomonoester moieties of a singletype are modified in a method of the invention. For example, a proteintarget can have one or more phosphotyrosines as a result ofphosphorylation by a tyrosine kinase. However, a protein target can havemore than one type of phosphomonoester moiety, for example, as a resultof phosphorylation by a kinase that phosphorylates multiple amino acids,such as a serine/threonine kinase, or as a result of phosphorylation bymultiple kinases. Various phosphomonoester moieties of proteins havebeen exemplified above with respect to the activity of kinase enzymes;however it will be understood that a protein can include aphosphomonoester moiety that was added by an in vitro synthetic chemicalmethod.

A target compound can be obtained from a natural source or syntheticsource. Exemplary natural sources include, but are not limited to acell, bodily fluid, tissue or other source such as those set forth infurther detail below. Those skilled in the art will know or be able toreadily determine methods for isolating a target compound from asynthetic or natural source using methods known in the art. For example,a protein target can be isolated using methods such as those describedin Scopes, Protein Purification 3^(rd) Ed., Springer Verlag, New York(1994) or Coligan et al., Current protocols in Protein Science, JohnWiley and Sons, Baltimore, Md. (2000), each of which is herebyincorporated by reference. In particular embodiments of the invention, acrude cell lysate containing a collection of target compounds, such asproteins, can be directly detected in a method of the invention withoutfurther isolation. Alternatively, one or more target compounds can befurther isolated from at least one other cellular component prior to usein a method described herein.

Any of a variety of methods known in the art can be used for in vitroproduction of a synthetic target compound. For example, a protein targetcan be synthesized using methods described in Goodman et al. (Eds.),Synthesis of Peptides and Peptidomimetics, Vol. E22a. Georg ThiemeVerlag, Stuttgart (2002), which is hereby incorporated by reference.Protein targets, whether obtained from a synthetic or natural source canbe isolated using known methods including, for example, liquid phaseextraction, precipitation, solid-phase extraction, chromatography,centrifugation or the like. Such methods are described, for example, inScopes, supra (1994) or Coligan et al., supra (2000).

A protein target used in accordance with the invention can be a fragmentof a larger protein. Thus, a portion of a larger protein that isbelieved to have a phosphomonoester moiety or that is believed to becapable of being phosphorylated can be obtained and used. A largerprotein can be fragmented to obtain a desired protein target, forexample, by using a protease or chemical cleavage method. Any of avariety of proteases or chemical cleavage methods known in the art canbe used including, for example, those described in Coligan et al., supra(2000) or Barrett et al., Handbook of Proteolytic Enzymes, Elsevier,Amsterdam, The Netherlands (2004), each of which is hereby incorporatedby reference. If desired, a fragment to be used as a protein target canbe isolated from at least one other fragment of the larger protein.However, fragments need not be isolated and can be used in a mixture ifdesired.

Fragmentation can be carried out prior to, during or after reaction ofthe protein target with a carbodiimide compound for protection of anelectrophile. Furthermore, fragmentation can be carried out prior to,during or after reaction of the protein target with a carbodiimidecompound and a nucleophile for modification of a phosphomonoestermoiety. In embodiments where the protein target is treated with akinase, phosphatase or other agent such treatment can be carried out,prior to, during or after fragmentation. A protein target can beattached to a solid-phase substrate, for example, via linkage to aphosphate moiety or other moiety, during fragmentation or fragmentationcan occur in solution-phase.

A protein target used in a method of the invention will have a primarystructure consisting of its sequence of amino acids. Typically, proteintargets will also have one or more secondary structure elements such asan alpha helix, or beta sheet. A sufficient number of amino acids canalso be present in a protein to allow formation of tertiary structure,also referred to as a protein fold. Furthermore, a target protein caninclude two or more amino acid chains that are associated according to aquaternary structure. A protein target used in the invention can be in anative state with respect to its primary, secondary, tertiary, and/orquaternary structure. However, if desired a target protein can betreated such that it loses native structure at one or more of the 4levels. For example, denaturation can be carried out to alterquaternary, tertiary or even secondary structure by the addition ofreagents such as urea, guanidinium salts, detergents, organic solvents;heating, mechanical disruption or other known methods.

Denaturation typically leads to loss of function for the protein target,thereby forming an inactivated protein target. A protein target can bedenatured or otherwise inactivated, for one or more reactions utilizedin a method of the invention. For example, an inactivated protein targetcan be treated with carbodiimide and a nucleophilic compound formodification of a phosphomonoester moiety of the protein target. Ifdesired, a target protein can also be in an inactivated state whenelectrophilic moieties such as carboxylic acids are protected by acarbodiimide compound. Inactivated protein targets can also be treatedwith one or more kinases or phosphatases in a method of the invention.Such inactivation, for example, when removing tertiary or quaternarystructure, can provide the advantage of exposing a phosphomonoestermoiety and/or carboxylic acid moiety that is sterically blocked from adesired reaction when in the protein target is in a native conformation.Alternatively, use of a protein target in a native conformation isdesirable for some embodiments of the invention, for example, whenpreferential modification of surface exposed moieties of the nativeprotein target is desired or when reaction of internal amino acids of anative protein target is to be avoided or reduced.

In particular embodiments, a target compound can be attached to a moietythat allows detection or fractionation of the target compound. Forexample, a target compound can be attached to a label moiety such as oneor more of the primary labels or secondary labels described herein. Aparticularly useful label is a nucleic acid moiety having a sequencethat can be used to identify the target compound to which it isattached. As demonstrated in Example I, the use of different nucleicacid labels each having a different nucleotide sequence and eachattached to a particular target compound in a plurality of differenttarget compounds, can allow the target compounds to be distinguishedaccording to their location on an array of complementary nucleic acidprobes. Similar array based detection of different target compounds canbe used with other label moieties as well.

A target protein can be attached to a nucleic acid moiety in any of avariety of configurations. In particular embodiments, a protein targetis attached to a nucleic acid moiety in a linear arrangement such thatthe nucleotide at the 3′ or 5′ end of the nucleic acid moiety isattached to the carboxy terminus or amino terminus of the proteintarget. As described in Example I, attachment of the 3′ end of a nucleicacid moiety to the amino terminus of a protein moiety provides a usefulconfiguration. Alternatively, the protein and nucleic acid can beattached such that an internal amino acid of the protein target isattached to the nucleic acid moiety and/or an internal nucleotide of thesequence of the nucleic acid moiety is attached to the protein target.Typically, the nucleic acid moiety, when used as a label, will not havea phosphomonoester moiety.

Attachment of a protein target and nucleic acid moiety is typicallymediated by at least one covalent bond. An attaching bond can be made toany desired portion of a protein target including, without limitation, abackbone carbon, nitrogen or oxygen or a sidechain (“R”) group.Similarly, a bond can be made to any desired portion of the nucleic acidmoiety including, but not limited to, the backbone or base. In theexemplary case where DNA or RNA is used, an attaching bond can be madeto the phosphodiester backbone, the sugar moiety or the base. If DNA orRNA analogs, such as those set forth above, are used then a covalentbond used for attachment can be made to known structural moietiestherein. Attachment of a PNA-based nucleic acid moiety to a proteinmoiety is particularly convenient since both moieties contain a similarbackbone structure. More specifically, the two moieties can be attachedvia a peptide bond between the terminal alpha carbonyl of one moiety andterminal alpha amino of the other moiety. PNA or peptide-PNA chimaerascan be synthesized using methods known in the art as described, forexample, in U.S. Pat. No. 6,713,602 or Nielsen et al. Science254:1497-1500 (1991), each of which is hereby incorporated by reference.

An exemplary method for attaching a protein moiety to a DNA moiety iscoupling a benzaldehyde residue on the DNA moiety with an aminooxyaceticon the amino terminus of the protein to form an oxime bond. Further,exemplary methods for attaching a DNA moiety to a protein moiety includecoupling a benzaldehyde residue on the DNA with a hydrazine on the aminoterminus of the protein to moiety to form a hydrazone bond, and othermethods described in U.S. Ser. No. 11/090,094, filed on Mar. 25, 2005,Zubin et al., Russian Chemical Reviews 71:239-264 (2002) or Tung et al.,Bioconjugate Chemistry 11:605-618 (2000), each of which is herebyincorporated by reference.

Attachment between a target compound and label moiety can be mediated bycovalent bonds, such as those exemplified above. Non-covalentinteractions can also mediate attachment between moieties. For example,each moiety can include a partner capable of forming a receptor-ligandcomplex such as avidin & biotin or other pairs set forth elsewhereherein or known in the art.

A target compound used in a method of the invention can be in solutionor attached to a solid-phase surface. Furthermore, the target compoundcan occur in solution-phase or solid-phase for different steps of amethod set forth herein. Exemplary embodiments are described in ExampleI, wherein a target protein-DNA conjugate having a phosphorylated aminoacid is contacted with a carbodiimide compound and a dye having an aminemoiety in solution phase. As further demonstrated in Example I, thedye-modified target can subsequently be attached to a solid-phasesubstrate via interaction of the DNA portion of the conjugate with acomplementary DNA probe on the solid-phase substrate. Several of theembodiments described in Example I also include pre-treatment of theDNA-protein conjugate with a carbodiimide compound in solution to form acarboxyl-protected DNA-protein conjugate.

In particular embodiments, a target compound that is attached to asolid-phase substrate can be contacted with a carbodiimide compound andan amine under conditions for addition of the amine to one or morephosphomonoesters of the target compound. If desired, the targetcompound can be pre-treated while attached to a solid-phase substrate,for example, by contacting the solid-phase-bound target compound to acarbodiimide compound under conditions for preferential addition of thecarbodiimide compound to a carboxylic acid moiety of the compound overthe phosphomonoester moiety of the compound, thereby forming acarboxyl-protected target compound.

In embodiments, including attachment of a target compound or other agentto a solid-phase substrate, particularly useful solid-phase substratesinclude, for example, magnetic beads which can be easily introduced to areaction mixture and easily removed with a magnet. Other known affinitychromatography substrates can be used as well. Known methods can be usedto attach a nucleic acid or protein moiety to a solid support including,for example, those described in U.S. patent application Ser. Nos.10/651,568 or 10/739,959, WO 01/41918, or WO 04/001646, each of which ishereby incorporated by reference. A solid support can be selected, forexample, from those materials described below with respect to detectionarrays.

A plurality of target compounds can be used in a method of theinvention. Example I demonstrates, evaluation of pools having 6, 96,100, or 194 target compounds. Further examples of pool sizes that can beused in the invention include, but are not limited to, those having atleast about 5, 10, 50, 100, 500, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶ or moretarget compounds. The target compounds in a pool can be treatedsimultaneously or sequentially in a method of the invention, such thatall or a subset of the target compounds are modified to have a protectedelectrophile moiety or a nucleophile-modified phosphate moiety or both.Furthermore, a method can be used in a multiplex format wherein a poolof several target compounds is treated with a pool of several differentagents such as a pool of kinases, phosphatases, or inhibitors. The poolof agents can include, for example, at least about 5, 10, 50, 100, 500,1×10³, 1×10⁴, 1×10⁵, 1×10⁶ or more different agents. A multiplexreaction can include any combination of these exemplary numbers oftarget compounds and other agents. Methods including use of such otheragents are set forth in further detail below and demonstrated in ExampleI.

Multiple target compounds or other agents used in accordance with theinvention can be attached to one or more solid-phase substrates. Otheragents include, without limitation, one or more kinases, phosphatases,receptors for secondary labels or other components of the methods setforth herein. One or more solid-phase substrates used in the inventioncan be in the form of an array. In particular embodiments, targetcompounds or other agents can be attached to particles that are arrayedor otherwise spatially distinguished. Exemplary particles includemicrospheres or beads. It will be understood that particles such asmicrospheres or beads can be spherical or approximately spherical butneed not be perfectly spherical. Rather, solid-phase particles havingother shapes including, but not limited to, cylinders, disks, plates,chips, slivers or irregular shapes can be used. In addition, particlesused in the invention can be porous having internal surfaces, thusincreasing the surface area available for attachment or detection oftarget compounds or other agents. Particle sizes can range, for example,from nanometers such as about 100 nm beads, to millimeters, such asabout 1 mm beads, with particles of intermediate size such as at mostabout 0.2 micron, 0.5 micron, 5 micron or 200 microns being useful.

The composition of particles useful in the invention can vary depending,for example, on the application of the invention or the method ofsynthesis. Typically, useful particles consist of a substantiallynon-compressible or inelastic material compared to a biological cell,examples of which include plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex, Teflon™, cross-linked dextranssuch as Sepharose™, cellulose, or nylon. However, if desired abiological cell or similarly compressible particle such as across-linked micelle can be used as a solid-phase support in theinvention. Other suitable particle compositions include, but are notlimited to, those used in peptide (protein), nucleic acid and organicmoiety synthesis or others described, for example, in MicrosphereDetection Guide, Bangs Laboratories, Fishers Ind., which is herebyincorporated by reference.

Exemplary bead-based arrays that can be used in the invention include,without limitation, those in which beads are associated with a solidsupport, examples of which are described in U.S. Pat. No. 6,355,431 B1,US 2002/0102578 and PCT Publication No. WO 00/63437, each of which ishereby incorporated by reference. A particularly useful solid support isa fiber optic bundle as described, for example, in U.S. Pat. No.6,200,737; WO 98/40726; or WO 98/50782, each of which is herebyincorporated by reference. Beads can be located at discrete locations,such as wells, on a solid-phase support, whereby each locationaccommodates a single bead. Alternatively, each discrete location caninclude a plurality of beads as described, for example, in US Pat. App.Pub. Nos. US 2004/0263923, US 2004/0233485, US 2004/0132205, or US2004/0125424, each of which is hereby incorporated by reference. It willbe understood that the sites of an array of the invention need not bediscrete sites. For example, it is possible to use a uniform surface ofadhesive or chemical functionalities that allows the attachment ofparticles at any position.

Beads or other particles can be loaded onto array supports using methodsknown in the art such as those described, for example, in U.S. Pat. No.6,355,431, which is hereby incorporated by reference. In someembodiments, particles can be randomly deposited on a solid-phasesubstrate. In embodiments where the placement of particles is random, acoding or decoding system can be used to localize and/or identify theparticles at each location in the array, thereby locating the targetcompound or other agent attached to the particle. This can be done inany of a variety of ways, for example, as described in U.S. Pat. No.6,355,431 or WO 03/002979, each of which is hereby incorporated byreference. A further coding system that is useful in the invention isthe use of diffraction gratings as described, for example, in US Pat.App. Nos. US 2004/0263923, US 2004/0233485, US 2004/0132205, or US2004/0125424, each of which is hereby incorporated by reference.Alternatively, particles can be attached to a support in a non-random orordered process.

An array of beads useful in the invention can also be in a fluid formatsuch as a fluid stream of a flow cytometer or similar device. Exemplaryformats that can be used in the invention to distinguish beads in afluid sample using microfluidic devices are described, for example, inU.S. Pat. No. 6,524,793, which is hereby incorporated by reference.Commercially available fluid formats for distinguishing particles suchas beads include, for example, those used in xMAP™ technologies fromLuminex or MPSS™ methods from Lynx Therapeutics.

Any of a variety of other arrays known in the art can be used in thepresent invention for attachment of target compounds or other agents.Commercially available microarrays that can be used in the inventioninclude, for example, an Affymetrix®GeneChip® microarray or othermicroarray synthesized in accordance with techniques sometimes referredto as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis)technologies as described, for example, in U.S. Pat. Nos. 5,324,633;5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716;5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974,164;5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269;6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752 and6,482,591, each of which is hereby incorporated by reference. A spottedmicroarray can also be used in a method of the invention. An exemplaryspotted microarray is a CodeLink™ Array available from AmershamBiosciences. Another microarray that is useful in the invention is onethat is manufactured using inkjet printing methods such as SurePrint™Technology available from Agilent Technologies. Other microarrays thatcan be used in the invention include, without limitation, thosedescribed in Butte, Nature Reviews Drug Discov. 1:951-60 (2002) or U.S.Pat. Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734;5,837,858; 5,919,523; 6,287,768; 6,287,776; 6,288,220; 6,297,006;6,291,193; and 6,514,751; and WO 93/17126; WO 95/35505, each of which ishereby incorporated by reference.

The surface of a solid-phase support can include a plurality ofindividual arrays that are physically separated from each other by anyof a variety of partitions. In particular embodiments, physicalseparation can be due to the presence of assay wells, such as in amicrotiter plate. Such a composite array (or array of arrays) can allowparallel processing of multiple samples in a method of the invention.For example, multiple samples each having a plurality of differenttarget compounds and/or other agents can be treated in parallel using acomposite array for multiplex detection of the phosphorylation states ofthe target compounds on each array. Exemplary composite arrays that canbe used in the invention are described in U.S. Pat. No. 6,429,027; WO02/00336 and U.S. Pat. App. Pub. No. 2002/0102578, each of which ishereby incorporated by reference.

A solid-phase support used in an array of the invention can include anymaterial that is capable of being attached to a desired target compoundor other agent. Useful supports include, but are not limited to, glass;modified glass; functionalized glass; plastics such as acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, or thelike; polysaccharides; nylon; nitrocellulose; resins; silica;silica-based materials such as silicon or modified silicon; carbon;metal; inorganic glass; optical fiber bundles, or any of a variety ofother polymers. Useful supports include those that allow opticaldetection, for example, by being translucent to energy of a desireddetection wavelength and/or by themselves not appreciably fluorescent atparticular detection wavelengths.

In particular embodiments, it is desired to detect phosphorylationstate, kinase activity or phosphatase activity using assays havingredundancy with regard to a particular target compound. Redundancy canbe particularly useful for increasing confidence levels or determiningstatistical validity for measurements such as kinetic or thermodynamicproperties of kinase or phosphatase activity including, for example,binding constants, maximum velocity, catalytic rate constant or othersdetermined using methods set forth in further detail below and in Segel,Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium andSteady-State Enzyme Systems, Wiley, John & Sons, Incorporated (1994),which is hereby incorporated by reference. Several other non-limitingadvantages of such redundancy, include the ability to make quantitativeestimates of confidence about collected data. Also redundancy canprovide substantial increases in sensitivity due to the ability to sumsignals from different target compounds. A variety of statisticalmathematical analyses can be done for analysis of large data sets.Exemplary analyses include, but are not limited to, baseline adjustment,averaging, standard deviation analysis, distribution and clusteranalysis, confidence interval analysis, mean testing, or the like, asdescribed in texts such as Freund and Walpole, Mathematical Statistics,Prentice Hall Inc., New Jersey (1980), which is hereby incorporated byreference. Methods for making and using redundant arrays are described,for example, in U.S. Pat. No. 6,355,431 and WO 00/60332, each of whichis hereby incorporated by reference.

A method of the invention can, optionally, include a step of contactinga target compound with a carbodiimide compound under conditions forpreferential addition of the carbodiimide compound to an electrophilemoiety, such as a carboxylic acid moiety, of the target compound,thereby forming an electrophile-protected target compound such as acarboxyl-protected target compound. Preferential addition of a reagentto a first moiety over a second moiety in a reaction can result inexclusive addition of the reagent to the first moiety. However,exclusive addition to a particular moiety is not necessarily required.Thus, preferential addition can result in a product having a largerproportion of the reagent attached to the first moiety than the amountof reagent attached to the second moiety. For example, in embodimentswhere phosphomonoester moieties of a target compound are to be modified,a population of target compounds can be pre-treated under conditions forpreferential addition of carbodiimide compound to electrophilic moietiesof the target compounds, wherein a lesser amount of carbodiimidecompound is added to the phosphomonoester moieties, so long as asufficient free phosphomonoester moieties are available for subsequentmodification.

An example of an electrophilic moiety that can be protected in a methodof the invention is a carboxylic acid moiety. A carbodiimide compoundcan be preferentially added to a carboxylic acid moiety over aphosphomonoester moiety at low pH. An example of the use of low pH forpreferential addition of a carbodiimide compound to a carboxylic acidmoiety is provided in Example I where pH 4.5 is used. The pH used in amethod of the invention can be at most about 3, 4, 5, 6, 7 or higherand/or at least about 6, 5, 4, 3, 2 or lower. In particular embodiments,the preferential addition of a carbodiimide compound to a carboxylicacid can be carried out at a pH in the range of 2.0 to 7.5.

The pH can be chosen to suit a particular target compound. For example,a protein target having acid-stable phospho-amino acids, such asphosphoserine, phosphothreonine or phosphotyrosine, can be subjected tolow pH for an extended period of time. Thus, the methods can be used toModify a phosphomonoester moiety of an acid-stable target compound suchas one or more acid-stable phospho-amino acids of a protein target.Acid-labile target compounds can also be used under appropriateconditions. For example, treatment of a target protein having anacid-labile phospho-amino acid, such as phosphohistidine, in order toprotect a carboxylic acid moiety, can be carried out at higher pH and/orfor shorter reaction times compared to the conditions used for a targetprotein having an acid-stable phosphomonoester moiety.

A possible mechanism for protecting a carboxyl moiety with acarbodiimide compound is provided in FIG. 1. This mechanism is providedfor purposes of illustration and is not necessarily intended to limitthe invention set forth herein. In this regard, it will be understoodthat a protected carbonyl made or used in accordance with thisdisclosure can have a structure other than those conjectured in FIG. 1.Nevertheless, as shown in FIG. 1, reaction of a carbodiimide compoundwith a carboxylic acid is believed to produce an O-acylurea, which issubsequently rearranged to form an N-acylurea. Accordingly, reaction ofa carbodiimide compound with a carboxylic acid moiety can be allowed toproceed under conditions for formation of either an O-acyl moiety or anN-acylurea moiety. As described in Example I, reaction at low pH for 90minutes was sufficient to protect carboxyl moieties of protein targets,presumably by formation of N-acylurea moieties. Any reaction timesufficient to protect a carboxyl moiety, for example, by formation of anN-acylurea moiety can be used.

A method of the invention can, optionally, include a step of contactinga target compound with a carbodiimide compound and a nucleophiliccompound, such as an amine, under conditions for addition of thenucleophilic compound to a phosphomonoester of the target compound. Thetarget compound can be, but need not be, an electrophile-protectedcompound such as a carboxyl-protected target compound.

In embodiments wherein a target compound is contacted with a firstcarbodiimide to form an electrophile-protected target compound and theelectrophile-protected target compound is contacted with a secondcarbodiimide under conditions for addition of a nucleophile to theelectrophile-protected target compound, the first and secondcarbodiimide compounds can be the same molecular species. An advantageof the methods as carried out in such embodiments is that the targetcompound need not be isolated from other reaction components between thereaction for electrophile-protection and the reaction for nucleophilicaddition (i.e. the method can be carried out in a one-pot reaction).Thus, not only can the same species of carbodiimide compound be used toactivate both reactions, but both can be added to the same reactionvessel. The first and second carbodiimide compounds can be added to thevessel simultaneously or sequentially, as desired. For example, anamount of carbodiimide sufficient to activate both reactions can beinitially added to a reaction vessel such that excess carbodiimidepresent after completion of the electrophile-protection reaction isavailable for the nucleophilic addition reaction. In this case, asubpopulation of the carbodiimide compounds can contact the electrophileto produce the electrophile-protected target compound and the excesscarbodiimide from this reaction can then contact the phosphomonoestermoiety for activation of the nucleophilic addition reaction. Continuingwith the example of a one pot reaction, the nucleophilic additionreaction can be initiated by addition of the nucleophile to the reactionvessel. The advantages set forth above can be extended to any of avariety of reaction vessels including, without limitation, a tube,multi-well plate, solid-phase surface, array or flow cell.

If desired, different molecular species of carbodiimide can be used forone or more reactions that occur in a method of the invention. Differentmolecular species of carbodiimide compound can be used, for example, incases where different species are known or believed to have differentefficiencies for each reaction. Again, the different molecular speciesof carbodiimide can be added to a reaction vessel simultaneously orsequentially.

Any of a variety of carbodiimide compounds capable of modifying acarboxylic acid moiety or activating phosphomonoester moiety fornucleophilic addition or both can be used in the invention. Exemplary,carbodiimide compounds include, but are not limited to,1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC);1-cyclohexyl-3(2-morpholinoethyl) carbodiimide-metho-p-toluenesulfonate; (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline);dicyclohexyl carbodiimide; diisopropyl carbodiimide or others known inthe art such as those described in Dolinnaya et al., Nucleic AcidResearch 19:3073-3080 (1991), which is incorporated herein by reference.By way of example, use of EDC for modification of carboxylic acidmoieties and activation of phosphomonoester moieties is set forth inExample I. The above carbodiimides or others known in the art can beused to activate other electrophilic moieties in a method of theinvention and can be selected for desired reactivity or othercharacteristics according to the particular application of the method.

Addition of a nucleophile to a carbodiimide-activated phosphomonoesterof a target compound using a method set forth herein can allow any of avariety of phosphomonoester-specific modifications of the targetcompound. In particular embodiments, the modification can be attachmentto a solid-phase substrate. For example, a nucleophilic compound, suchas an amine, that is reacted with a target compound can be attached to asolid-phase substrate. Accordingly, following activation of aphosphomonoester moiety of the target compound with a carbodiimidecompound and addition of the solid-phase bound amine, the targetcompound will be attached to the solid-phase via the phosphate moiety.The solid-phase substrate can be any of those set forth above in regardto attachment of a target compound including, for example, a particle orarray solid-phase substrate.

A nucleophilic compound can be attached to a solid-phase substrate byreaction with any of a variety of linkers having a phosphomonoesterreactive moiety. Solid-phase substrates having phosphomonoester reactivemoieties can be obtained from commercial suppliers such as BangsLaboratories (Fishers, Ind.), Pierce Chemicals (Rockford, Ill.) orSigma-Aldrich (St. Louis, Mo.). The linkage between a solid-phase andphosphomonoester moiety of a target compound can be polymeric, forexample, including a polypeptide or polynucleotide sequence, hydrophobicor hydrophilic. A linker can further include a cleavable moiety,examples of which include a moiety, such as ethylene glycol, that iscleavable by hydroxylamine; a moiety, such as a sulfone, that iscleavable by base; a moiety, such as a disulfide, that is cleavable byreducing agent such as a thiol; or a moiety, such as tartarate, that iscleavable by an oxidizing agent such as periodate. Other exemplaryattachments include products of a reaction of a crosslinking agent, suchas those set forth below, with a surface having a moiety that isreactive toward the crosslinking agent.

A method of the invention can be used to add a reactive moiety to atarget compound. For example, a nucleophilic compound can be acrosslinking agent having a first reactive moiety that is reactive withphosphomonoester and a second reactive moiety. A particularly usefulcross-linking agent is a heterobifunctional crosslinking agent in whichthe first and second reactive moieties are different species. Typically,the second reactive moiety will be inert to activation with carbodiimideand reaction with phosphomonoester. However, a crosslinking agent inwhich both moieties are reactive toward phosphomonoester, such as ahomobifunctional crosslinking agent, can be used. Use of a crosslinkingagent in which both moieties are reactive toward phosphomonoester can beused to crosslink two phosphate moieties. Alternatively or additionally,one of the moieties can have a protecting group that can be removedfollowing nucleophilic addition of the crosslinking agent to a phosphatemoiety of a target compound. Furthermore, a crosslinking agent caninclude a cleavable linker such as those set forth above.

Examples of phosphomonoester reactive moieties that can be included in acrosslinking agent include, but are not limited to, an amine, hydrazine,sulfhydril or hydroxyl. The second moiety of a crosslinking agent can bereactive to phosphomonoester and/or other moieties. Examples include,but are not limited to, a second moiety that is reactive towardcarboxylic acid, such as an amine moiety; a second moiety that isphotoreactive such as a nitrophenyl azide or hydroxyphenyl azide moiety;a second moiety that is reactive toward an amine moiety such as acarboxylic acid, NHS-ester or imidoester; or a second moiety that isreactive toward sulfhydril, such as a maleimide, pyridyldisulfide,pyridyldithio, bromoacytal or iodoacytal. Another example of a usefullinker is cysteamine, which has an amino moiety that can be added to aphosphomonoester moiety as described herein, and an internal disulfidethat can be subsequently reduced to generate a reactive free sulfhydrilmoiety. Crosslinking agents can be obtained from commercial supplierssuch as Pierce Chemicals (Rockford, Ill.) or Sigma-Aldrich (St. Louis,Mo.).

In particular embodiments, a label moiety can be attached to thephosphomonoester in a method of the invention. For example, anucleophile, such as an amine, that is added to a carbodiimide-activatedphosphomonoester can include a label moiety. Modification to add a labelto a phosphorylated target compound is demonstrated in Example I.Exemplary label moieties include, but are not limited to primary andsecondary labels set forth previously herein. Detection of the labelmoiety can be used to determine presence of a phosphomonoester moiety ina target compound.

Accordingly, the invention further provides a method of detecting aphosphomonoester moiety of a target compound. The method can include thesteps of (a) providing a target compound having an electrophilic moiety,such as a carboxylic acid moiety, and a phosphomonoester moiety; (b)contacting the target compound with a first carbodiimide compound underconditions for preferential addition of the first carbodiimide compoundto the electrophilic moiety over the phosphomonoester moiety, therebyforming an electrophile-protected target compound; (c) contacting theelectrophile-protected target compound with a second carbodiimidecompound and a nucleophilic compound, such as an amine, under conditionsfor addition of the nucleophilic compound to the phosphomonoestermoiety, thereby forming a nucleophile-modified phosphate moiety; and (d)detecting the nucleophile-modified phosphate moiety.

A nucleophilic compound that is added to a phosphate moiety can bedetected due to the presence of a label moiety. Useful properties of alabel moiety include, those that can be used to distinguish differenttarget compounds alone, or in combination with other methods, such asattachment of the target compounds to solid-phase supports. Exemplaryproperties upon which detection can be based include, but are notlimited to, mass, electrical conductivity, energy absorbance,fluorescence, magnetism, luminescence or the like.

Detection of fluorescence can be carried out by irradiating a targetcompound or label moiety with an excitatory wavelength of radiation anddetecting emitted radiation by methods known in the art and describedfor example in Lakowicz, Principles of Fluorescence Spectroscopy, 2ndEd., Plenum Press New York (1999), which is hereby incorporated byreference. A fluorophore can be detected based on any of a variety offluorescence phenomena including, for example, emission, excitation,fluorescence resonance energy transfer (FRET) intensity, quenching,anisotropy or lifetime at one or more wavelengths.

Other detection techniques that can be used to detect a target compoundinclude, for example, mass spectrometry which can be used to perceive amolecule or complex based on its mass; surface plasmon resonance whichcan be used to perceive a molecule or complex based on binding ordissociation from a surface; absorbance spectroscopy which can be usedto perceive a molecule or complex based on the wavelength of the energyit absorbs; calorimetry which can be used to perceive a molecule orcomplex based on changes in temperature of its environment upon bindingor dissociation; electrical conductance or impedance which can be usedto perceive a molecule or complex based on changes in its electricalproperties or in the electrical properties of its environment; magneticresonance which can be used to perceive a molecule or complex based onpresence of magnetic nuclei; or other known analytic spectroscopic orchromatographic techniques.

A label moiety can impart a target compound with a characteristic thatallows its separation from other components of a sample. For example,the nucleophilic compound can include a ligand that is useful foraffinity chromatography or solid-phase extraction of a target compoundusing a solid-phase having a receptor for the ligand. Alternatively oradditionally, the label moiety can be used to track the modified targetcompound in a separation method including, for example, achromatographic separation such as reverse-phase, normal phase, ionexchange, or size exclusion chromatography; extraction, such assolid-phase or liquid-liquid phase extraction; precipitation; gelelectrophoresis; capillary electrophoresis; differential centrifugation;flow cytometry, or mass spectroscopy.

A method of the invention can further include a step of identifying thetype of amino acid that is attached to a phosphomonoester moiety of aprotein. The type of amino acid can be, for example, a serine,threonine, tyrosine, histidine, glutamic acid or aspartic acid. The typeof amino acid can be identified based on the known sequence of theprotein. For example, in the case of a short protein, such as a fragmentfrom a larger protein, wherein the fragment has only a single amino acidthat is capable of being phosphorylated under the conditions tested, theidentity of the modified phosphate can be inferred from the compositionand/or sequence of the fragment. Alternatively, a phospho-amino acid canbe identified using an analytical method including, for example, achromatographic technique such as reverse-phase HPLC or thin layerchromatography or a spectroscopic technique such as mass spectroscopy ornuclear magnetic resonance spectroscopy. Additionally, amino acidanalyzers available from commercial sources can be used such as thePerkin Elmer Applied Biosystems Model 140C PTH Amino Acid Analyzer. Suchmethods can be carried out on an intact protein, or followingfragmentation of the protein, for example, via chemical methods such asacid-hydrolysis and/or base hydrolysis or via enzymatic methods such asprotease hydrolysis.

Furthermore, the location of the phosphorylated amino acid in theprimary sequence of the protein can be determined in a method of theinvention. The location can be inferred from the known sequence of theprotein, for example, in combination with one or more analyticaltechnique for identifying the type of amino acids present in a protein,such as those set forth above. If desired a protein sequencing methodcan be used such as a method based on Edman degradation and relatedchemistry. Protein sequencing instruments available from commercialsources can be used such as the Perkin Elmer Applied Biosystems Model494 Procise protein/peptide sequencer or the Perkin Elmer QSTAR hybridLC/MS/MS mass spectrometer.

A method of the invention can include a step of contacting a targetcompound with a kinase under conditions for adding a phosphate moiety tothe target compound. For example, a protein target can be contacted witha protein kinase prior to treating the protein target with acarbodiimide and amine. Thus, addition of the amine can be detected asan indication of activity of the kinase toward the protein target. Anyof a variety of known kinases can be used including, for example, aprotein kinase (i.e. a kinase that adds a phosphate to a proteinsubstrate), adenylate kinase, creatine kinase, pyruvate kinase,hexokinase, nucleotide diphosphate kinase or thymidine kinase. Exemplaryprotein kinases that are useful in the invention include, but are notlimited to, a serine/threonine kinase, tyrosine kinase, histidinekinase, or aspartic acid/glutamic acid kinase. A kinase useful in theinvention can be from either a prokaryote or eukaryote, including eithera plant or animal.

A kinase can belong to any of the following families of proteins: cyclicnucleotide regulated protein kinase (PKA & PKG) family;diacylglycerol-activated/phospholipid-dependent protein kinase C (PKC)family; kinases that phosphorylate G protein-coupled receptors family;budding yeast AGC-related protein kinase family; kinases thatphosphorylate ribosomal protein S6 family; budding yeast DBF2/20 family;flowering plant PVPK1 protein kinase homolog family; kinases regulatedby Ca2+/CaM and close relatives family; KIN1/SNF1/Nim1 family;cyclin-dependent kinases (CDKs) and close relatives family; ERK (MAP)kinase family; glycogen synthase kinase 3 (GSK3) family; casein kinaseII family; Clk family; Src family; Tec/Atk family; Csk family; Fes (Fps)family; Abl family; Syk/ZAP70 family; Tyk2/Jak1 family; Ack family;focal adhesion kinase (Fak) family; epidermal growth factor receptorfamily; Eph/Elk/Eck receptor family; Axl family; Tie/Tek family;platelet-derived growth factor receptor family; fibroblast growth factorreceptor family; insulin receptor family; LTK/ALK family; Ros/Sevenlessfamily; Trk/Ror family; DDR/TKT family; hepatocyte growth factorreceptor family, nematode Kin15/16 family; Polo family; MEK/STE7 family;PAK/STE20 family; MEKK/STE11 family; NimA family; wee1/mik1 family;kinases involved in transcriptional control family; Raf family;activin/TGFb receptor family; flowering plant putative receptor kinasesand close relatives family; PSK/PTK “mixed lineage” leucine zipperdomain family; casein kinase I family; and PKN prokaryotic proteinkinase family.

Other known kinases that can be used include, for example, those setforth in Manning et al., Science 298:1912-1934 (2002); The proteinkinase resource web page (kinasenet.org, administered by The San DiegoSupercomputer Center and The University of California San Diego, withfunds from the National Science Foundation), each of which is herebyincorporated by reference. Variants of known kinases can also be usedincluding, for example, those known or suspected of being associatedwith a disease or condition, such as the kinase variants set forth atthe KinMutBase (see Ortutay et al., Hum Mutat. 25:435-42 (2005)), whichis hereby incorporated by reference.

A method of the invention can include a step of contacting aphosphorylated target compound with a phosphatase under conditions forremoving a phosphate moiety from the target compound. For example, aphospho-protein target can be contacted with a phosphatase prior totreating the protein target with a carbodiimide and amine. Thus,inability of the amine to add to the target protein can be detected asan indication of activity of the phosphatase toward the phospho-proteintarget. The inability of the amine to add to the protein can bedetermined, for example, by comparison of the amount of amine on thephosphatase treated phospho-protein target to the amount of amine on thesame species of phospho-protein target that has not been treated withthe phosphatase. Any of a variety of known phosphatases can be usedincluding, for example, a tyrosine phosphatase (PTP), serine/threoninephosphatase, metalloenzyme phosphatase, non-metalloenzyme phosphatase,cysteine phosphatase, dual-specificity phosphatase (DSPs); Cdc25phosphatase, myotubularin-related phosphatase, low molecular weightphosphatase, inositol 4-phosphatase or Sac1-domain phosphatase. Otherknown phosphatases that can be used include, for example, those setforth in Andersen et al., Mol. Cell. Biol. 21:7117-36 (2001), which ishereby incorporated by reference. Variants of known phosphatases canalso be used including, for example, those known or suspected of beingassociated with a disease or condition.

Methods including a step of contacting a target compound with a kinaseor phosphatase can be used to identify substrate specificity for thekinase or phosphatase, variations in kinase or phosphatase activity dueto presence of an activator or inhibitor, variations in kinase orphosphatase structure that result in altered activity, or the like. Insuch embodiments, the target compound can be a kinase or phosphatasesubstrate. Furthermore, a method of the invention can be used todetermine a kinase or phosphatase activity that is indicative of thepresence of the kinase or phosphatase in a sample. For example, a kinaseor phosphatase substrate having a known or determinable amino acidsequence can be contacted with a sample in a method of the invention andphosphorylation state of the substrate detected. Thus, the methods canbe used for discovery of a kinase or phosphatase in a biological samplewherein its presence and/or activity were previously not known. Suchmethods can be used for diagnosis or prognosis of a disease or conditionas set forth in further detail below.

A method of the invention can be used to characterize a biologicalsample such as a cell, tissue, organism, group of organisms, celllysate, tissue homogenate, or fraction thereof. For example, thephosphorylation state of one or more proteins in a first system can bedetermined and, if desired, can be compared to the phosphorylation stateof the one or more proteins of a second biological sample. Alternativelyor additionally, a phosphorylation state determined for a biologicalsample can be compared to a phosphorylation pattern that is expected orpredicted. It will be understood that, in this regard, thephosphorylation state for one or more proteins in a biological samplecan be due to the activity of one or more different kinases orphosphatases. Accordingly, the invention can be used to identify thecomplement of active kinases or phosphatases in a particular biologicalsample. For example, a biological sample can produce a particularsignature of phosphorylated protein or proteins that indicates thecomplement of active kinases or phosphatases.

If desired one or more kinase, phosphatase or target protein can beisolated from a biological sample for use in a method of the invention,for example, using methods set forth previously herein. However, abiological sample used in a method described herein, can be a complexmixture of kinases, phosphatases or target proteins. Thus, the methodscan be carried out in a multiplex fashion with respect to havingmultiple kinases, phosphatases, target proteins or a combinationthereof. For example, multiple kinases and/or phosphatases can becontacted with multiple target compounds in a multiplex format. In sucha case, modification of the target proteins can be detected using anarray to differentiate individual target proteins from each other, forexample, using methods exemplified in Example I.

As a further example, a method of the invention can be used toseparately evaluate the kinase or phosphatase activity of a biologicalsample toward one or more individual target substrates (i.e. target of akinase and/or phosphatase). More specifically, one or more targetcompounds can be contacted with a biological sample, or fractionthereof, under conditions in which one or more kinases are capable ofphosphorylating their substrates. The kinase activity can then bedetermined based on detection of the phosphorylation state of one ormore of the target compounds. Similarly a phosphorylated target compoundcan be added to a biological sample under conditions in which one ormore phosphatases is capable of removing the phosphate.

The ability to perform a multiplexed kinase and/or phosphatase assays inaccordance with the invention provides an advantage for the evaluationof signal transduction cascades or other biochemical pathways that areinfluenced by multiple kinases and/or phosphatases. A multiplexed assaycan more closely mimic the complexities of biological systems such thatevaluation of the results can yield observations and information that isdifferent or absent when the results of individual assays, using thecomponents of the multiplexed assay, are evaluated alone.

A method of the invention can be used to separately evaluate the effectof an inhibitor or activator on the kinase or phosphatase activity of abiological sample. More specifically, one or more target compounds canbe contacted with a biological sample, or fraction thereof, underconditions in which one or more kinases are capable of phosphorylatingtheir substrates. A similar sample can be prepared that additionallyincludes an agent that is a known inhibitor (or activator) or candidateinhibitor (or activator) of at least one of the kinases. The kinaseactivity can then be determined in both samples based on detection ofthe phosphorylation state of one or more of the target compounds. Theeffects of a known or candidate inhibitor (or activator) of aphosphatases can be determined by a similar sample comparison.

An activity of a kinase or phosphatase determined in a method of theinvention can include one or more characteristic. For example, the aminoacid sequence of a protein target that is phosphorylated ordephosphorylated can be determined and, if desired, the location wherephosphorylation or dephosphorylation occurs can also be identified. Suchevaluations can be used to determine specificity of a kinase orphosphatase for a particular amino acid sequence. The amino acidrecognition sequence identified in a method of the invention can be adiscreet sequence or a consensus sequence, having one or more degenerateposition. In embodiments employing multiple target proteins, the targetproteins can have different amino acid sequences such thatidentification of those sequences that are modified by a kinase orphosphatase, those that are not substantially modified by the kinase orphosphatase, or both can be used to determine the sequence specificityof the kinase or phosphatase.

Activity of a kinase or phosphatase can be determined qualitatively orquantitatively in a method of the invention. Exemplary qualitativedeterminations can include, without limitation, identification ofsubstrate specificity; identification of conditions that increase ordecrease activity; determination of the presence or absence ofactivators or inhibitors of the kinase or phosphatase; or determinationof relative activities between different samples, kinases, phosphatases,target compounds or conditions. Quantitative determinations can be madefor these or other characteristics for a more precise measure, ifdesired. For example, a method of the invention can be used to determinethe binding affinity of a kinase or phosphatase for a particular targetcompound in the form of a thermodynamic constant, such as a dissociationconstant. Similarly, an inhibition constant can be determined for aparticular inhibitor in the presence of a phosphatase and its targetsubstrate or in the presence of a kinase and its target substrate. Afurther quantitative measure that can be determined in a method of theinvention is a catalytic rate constant for phosphorylation or removal ofa phosphate. Such kinetic and thermodynamic constants can be determinedusing titration measurements and/or time dependent measurements inaccordance with analyses known in the art as described, for example, inSegel, supra (1994), which is hereby incorporated by reference.

Exemplary biological samples that can be used in a method of theinvention can include or be derived from a mammal such as a rodent,mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow,cat, dog, primate, human or non-human primate; a plant such asArabidopsis thaliana, corn (Zea mays), sorghum, oat (oryza saliva),wheat, rice, canola, or soybean; an algae such as Chlamydomonasreinhardtii; a nematode such as Caenorhabditis elegans; an insect suchas Drosophila melanogaster, mosquito, fruit fly, honey bee or spider; afish such as zebrafish (Danio rerio); a reptile; an amphibian such as afrog or Xenopus laevis; a dictyostelium discoideum; a fungi such aspneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a plasmodium falciparum. Amethod of the invention can also be used to detect phosphorylationstate, phosphatase activity or kinase activity for a prokaryote such asa bacterium, Escherichia coli, staphylococci or mycoplasma pneumoniae;an archae; a virus such as Hepatitis C virus or human immunodeficiencyvirus; or a viroid. A homogeneous culture or population of the abovesystems can be evaluated using the invention as can a collection ofseveral different organisms, for example, in a community or ecosystem.

A cell from which one or more target compounds, phosphatases or kinasesis obtained for use in the invention can be a normal cell or a celldisplaying one or more symptom of a particular disease or condition.Thus, a target compound, phosphatase or kinase used in a method of theinvention can be obtained from a cancer cell, neoplastic cell, necroticcell, cell experiencing an auto-immune condition, apoptotic cell or thelike. Those skilled in the art will know or be able to readily determinemethods for isolating one or more target compounds, phosphatases orkinases from a cell, bodily fluid or tissue using methods known in theart such as those described above in regard to isolation of proteintargets.

A method of the invention can further include steps of isolating aparticular type of cell or tissue. Exemplary methods that can be used ina method of the invention to isolate a particular cell from other cellsin a population include, but are not limited to, Fluorescent ActivatedCell Sorting (FACS) as described, for example, in Shapiro, PracticalFlow Cytometry, 3rd edition Wiley-Liss; (1995) (which is herebyincorporated by reference), density gradient centrifugation, or manualseparation using micromanipulation methods with microscope assistance.Exemplary cell separation devices that are useful in the inventioninclude, without limitation, a Beckman JE6™ centrifugal elutriationsystem, Beckman Coulter EPICS ALTRA™ computer-controlled FlowCytometer-cell sorter, Modular Flow Cytometer™ from Cytomation, Inc.,Coulter Counter™ or Channelyzer™ system, density gradient apparatus,Cytocentrifuge, Beckman J-6™ centrifuge, EPICS V™ dual laser cellsorter, or EPICS PROFILE™ flow cytometer. A tissue or population ofcells can also be removed by surgical techniques. For example, a tumoror cells from a tumor can be removed from a tissue by surgical methods,or conversely non-cancerous cells can be removed from the vicinity of atumor.

The invention can be used for diagnosis or prognosis of a disease orcondition. For example, the phosphorylation state, phosphatase activityor kinase activity for a test cell or tissue that is known or suspectedof being affected by a particular disease or condition can be determinedusing a method of the invention. If desired, phosphorylation state,phosphatase activity or kinase activity can also be determined for asecond cell or tissue that serves as a control and the results from thecontrol cell or tissue compared to the results from the test cell ortissue. A control cell or tissue can be derived from a non-affected cellor tissue from the same individual as the test cell or tissue.Alternatively, the control cell or tissue can be obtained from aseparate individual. The separate individual can be a non-affectedindividual that is related to the test individual within one, two, threeor more generations. Alternatively, the separate individual can beeffectively unrelated being many generations removed, or even of adifferent ethnicity. In some cases it may be useful to use a controlindividual having similar ethnicity as the test individual.

Phosphorylation state, phosphatase activity or kinase activitydetermined in a method of the invention for a particular biologicalsample can be correlated with one or more symptoms of a disease orcondition. Those skilled in the art will know or be able to determinesymptoms that are indicative of a disease or condition being evaluated.An exemplary reference describing symptoms for particular diseases orconditions is The Merck Manual of Diagnosis and Therapy 16th Ed., Editedby Berkow, published by Merck and Co., Inc., Rahway N.J. (1992), whichis hereby incorporated by reference.

A method of the invention can also be used to evaluate the effect of aparticular treatment on a biological sample. The biological sample canbe a cell experiencing a disease state or condition for which removal ofone or more symptoms by the treatment is desired. Alternatively, thebiological sample can be a normal cell for which adverse response to aparticular treatment is not desired, as it represents an off-targetresponse for the treatment. In particular embodiments, the treatment canbe administration of a drug or drug candidate to a biological sample,wherein the drug or candidate drug is suspected of having an effect onthe system. For example, following administration of the drug orcandidate drug the phosphorylation state, phosphatase activity or kinaseactivity can be measured for the treated system. Thus, the inventionprovides a method for screening drugs or drug candidates.

A drug or drug candidate used in a method of the invention can act, forexample, as a kinase inhibitor or phosphatase inhibitor, can act toincrease expression of a kinase or phosphatase, or can act todestabilize one or more kinase or phosphatase, thereby reducing halflife in the natural milieu. Comparison can be made to thephosphorylation state, phosphatase activity or kinase activity for acontrol system that has not been treated or that has been treated to adifferent extent. Thus, phosphorylation state, phosphatase activity orkinase activity, as measured by a method of the invention, can be usedto evaluate dose response, efficacy, time period of response or the likefor a biological sample undergoing a particular treatment. If available,comparison can be made to a reference activity, for example, as storedin a database or other storage medium.

The invention provides a protein having an N-acylurea moiety and aphosphoric amide moiety. Further provided is a plurality of proteinseach having an N-acylurea moiety and a phosphoric amide moiety. Theprotein can be made for example, according to a method set forth herein.The N-acylurea moiety can include the alpha carbonyl moiety of theC-terminal amino acid of the protein, the beta carbonyl moiety of anaspartate residue or the gamma carbonyl moiety of a glutamate residue.The phosphoric amide moiety can include a label moiety, cross-linkerhaving a reactive moiety or a linker attached to a solid-phasesubstrate. Thus, the protein is useful for an analytical method toidentify a characteristic of the protein or a characteristic of abiological sample from which it is derived.

It will be understood that the methods exemplified above with regard toadding a nucleophilic compound to a phosphomonoester can be used to adda nucleophilic compound to other phosphorus containing moieties such asa phosphoamide or thiophosphate. Generally, the methods can be used fornucleophilic addition to a variety of electrophilic moieties that areactivated by a carbodiimide compound. Furthermore, it will be understoodthat an electrophile-protected compound produced using a method setforth herein can be used in any of a variety of nucleophilic additionreactions, whether or not the nucleophilic addition reactions areactivated by a carbodiimide compound.

The following example is intended to illustrate but not limit thepresent invention.

Example I Chemical Labeling and Detection of Phosphorylated BiologicalMolecules

This example demonstrates a universal chemical approach forphosphorylation detection. This example further demonstrates analysis ofkinase activity in a multiplex format. This example also demonstratesuse of pattern recognition as a robust tool for studying kinase activityin a multiplex assay.

Assay Design

A solution-based kinase assay format was devised to demonstratemultiplexed kinase profiling on the Sentrix® BeadArray platform asschematically represented in FIG. 2. In order to make the assaycompatible with the Sentrix® BeadArray readout, protein (also referredto as “peptide”) substrates for the kinases were modified intoDNA-conjugated protein targets. Each different protein sequence wasconjugated to a unique DNA sequence and each DNA sequence wascomplementary to a unique DNA probe of the array. Thus, followinghybridization of the DNA-conjugated proteins to the array, each proteintarget can be identified according to its location on the array.

As shown in FIG. 2, a kinase recognizes the protein portion of aDNA-protein conjugate and phosphorylates the protein at a specific sitein the amino acid sequence resulting in a phosphorylated protein, insolution. A label molecule (fluorescent dye or biotin) containing anamino moiety is then reacted with the phosphomonoester moiety of theprotein, also in solution. The DNA-protein conjugate is incubated withthe Sentrix® BeadArray and the DNA moiety is hybridized to itscomplementary DNA on the array. Presence or absence of signal from thelabel at each probe location is detected. The presence of signal at aspecific probe location indicates that the respective protein moietyconjugated to the probe complement is phosphorylated.

Phosphorylated proteins were detected using the universal chemicalapproach shown in FIG. 1. As shown in FIG. 1B, a dye label having anamino moiety can be covalently attached to a phosphate moiety byactivating nucleophilic attack of the amine on the phosphomonoester with1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, Fluka) at pH 7.5.However, carboxylic moieties are also reactive under these conditions.As shown in FIG. 1A, an EDC molecule adds to the carbonyl to form anO-acylurea derivative that is subject to nucleophilic attack by theamino-containing dye when activated by a second EDC molecule. This canresult in unwanted labeling of a protein target at free carbonyls ofaspartic acid, glutamic acid and protein c-termini. Carbonyls wereblocked (i.e. protected) from subsequent dye-labeling by pre-treatingthe protein with EDC at pH 4.5 for 90 mins. The pre-treatment isbelieved to form O-acyl derivatives at carbonyls which rearrange to formstable N-acylurea derivatives of the carbonyls, as shown in FIG. 1A andas described in Toniolo et al., Helvetica Chimica Acta, 73:626 (1990),which is hereby incorporated by reference. Following pretreatment, theprotein can be reacted with EDC and the amino containing dye at pH 7.5such that N-acylated carbonyls are not reactive to amino-containing dye(as shown in FIG. 1A) and phosphorylated amino acids are selectivelylabeled (as shown in FIG. 1B).

Synthesis of Protein-DNA Conjugates

A DNA molecule having a benzaldehyde nucleotide at the 3′ end wassynthesized using methods described in U.S. Ser. No. 10/739,959, whichis hereby incorporated by reference. The benzaldehyde residue on the DNAwas coupled to an aminooxyacetic moiety on the amino terminus of aprotein, at pH between 4.0 and 5.5, to form an oxime bond as describedin U.S. Ser. No. 11/090,904, which is hereby incorporated by reference.

Specificity of Phosphate Detection Using Chemical Labeling

FIG. 3 shows the results of dye-labeling of proteins with and withoutEDC pre-treatment. R1 was a protein target containing a tyrosineresidue, R2 had the same sequence as R1, however, the tyrosine residuewas phosphorylated synthetically; R3 was a protein target containing aserine residue; R4 had the same sequence as R3, however, the serineresidue was phosphorylated synthetically; R5 was a protein targetcontaining an aspartic acid residue; R6 had a glutamic acid residue.

As shown in FIG. 3, when the proteins were reacted with amino-containingdye and EDC at pH 7.5, following pretreatment at pH 7.5 without EDC(identified as “pH 7.5” in the Figure) the R5 and R6 targets, whichcontained carbonyl moieties but no phosphate moieties were labeled alongwith the phosphorylated targets R2 and R4. Similar reactivity of freecarbonyls resulted when the proteins were pretreated with EDC at pH 7.5(labeled “pH 7.5, EDC pretreatment” in the Figure) or when the proteinswere pretreated at pH 4.5 in the absence of EDC (“pH 4.5”). However,when the proteins were pretreated with EDC at pH 4.5, targets R2 and R4,having phosphomonoester, were labeled, whereas targets R5 and R6, havingcarbonyls but no phosphomonoesters were blocked from being labeled. Thedata demonstrates that specific detection of phosphorylated protein wasachieved using low pH, EDC pre-treatment followed by EDC activatedamino-dye labeling at neutral pH.

Correlation of Antibody Labeling and Chemical Labeling Methods

A kinase profiling assay was carried out in four major steps, includingthe phosphorylation of protein target in a kinase enzyme reaction,labeling of phosphorylated target, hybridization of labeled target tothe array, and detection of hybridized target on the array.

In the first step, phosphorylation was carried out in duplex format suchthat a mixture of two DNA-protein conjugate targets, one having an aminoacid recognition sequence for p60c-src kinase and the other for PKAkinase, were co-incubated with p60c-src kinase and PKA kinase. Thereaction was performed in 15 ul of 50 mM HEPES, containing 0.1 mM EDTA,0.015% Brig 35, 0.15 mM ATP, 30 mM MgCl2, 0.1% 2-mercaptoethanol, pH7.5.

Upon completion of the enzymatic reaction; the second step was carriedout to detect phosphorylation using chemical labeling or antibody-basedlabeling. In the chemical labeling method, a solution of5-(aminoacetamido)fluorescein (NH2-FAM, Molecular Probes, OR) and EDC inMES buffer, pH 6.0, was added to the reaction mixture to a finalconcentration of 150 mM EDC and 200 μM of NH2-FAM. Where pre-treatmentwas used to block (i.e. protect) carbonyl moieties, the pH of thereaction mixture was first adjusted to pH 4.5 and then pre-treated with100 mM of EDC for 1.5 hours at 25° C. After the pre-treatment, theNH2-FAM and EDC were added and the reaction was allowed to proceedovernight. Where antibodies were used as detection reagents forphosphotyrosine, a mouse anti-phosphotyrosine-biotin conjugate (UpstateCell Signaling Solutions, VA) was used along with a streptavidin,R-phycoerythrin conjugate (Molecular Probes, OR). For phosphoserinedetection, five different biotinylated antibodies were tested each incombination with a streptavidin, R-phycoerythrin conjugate.

In the third step, the mixture from the labeling step was diluted intoGoldenGate™ hybridization buffer (Illumina, Inc., San Diego, Calif.) toa final concentration of 100 pM DNA-protein conjugate target andhybridized onto a Sentrix® BeadArray Matrix for 24 hours at roomtemperature. The BeadArray matrix included nucleic acid probes that werecomplementary to the DNA portion of the DNA-protein conjugate targets.

In the fourth step, the array was washed and then imaged using the UltraScanner (Illumina, Inc., San Diego, Calif.) set at 485 nm excitation and535 nm emission to detect the presence of fluorescein on the probes.

Seven different duplex assays were carried out, each with differentp60c-src and PKA kinase concentrations. Following the phosphorylationstep, each solution was divided into two samples for phosphorylationdetection. The first detection sample was evaluated using the EDCchemical detection method (FIG. 4A), the other was evaluated usinganti-phosphotyrosine and anti-phosphoserine antibodies (FIG. 4B). Asshown in FIG. 4A, a saturable increase in kinase activity was observedwhen the chemical labeling method was used to monitor kinase titrationof either kinase. A similar saturable response was observed using ananti-phosphotyrosine antibody to detect the p60c-src kinase substrate(which is expected to be phosphorylated at a tyrosine residue by thep60c-src kinase). An 89% correlation between the antibody based and theuniversal chemical detection methods was achieved for detection ofp60c-src kinase activity. The anti-phosphoserine antibody failed todetect the phosphorylation of the PKA substrate that was detected usingthe chemical labeling method.

Kinase Profiling Assay Results Using the p60c-src and PKA Systems

The kinase profiling assay was carried out using the four step method asset forth above with the following modifications. The enzymatic step wasperformed in solution using nanomolar amounts of kinase enzyme andmultiplexed protein target pools having 6, 96, 100, or 194 differentprotein targets. Upon completion of the enzymatic reaction, the chemicallabeling method was used, including low pH, EDC pre-treatment, to detectboth phosphotyrosine and phosphoserine residues.

For the inhibition studies, Src Kinase Inhibitor I and Protein Kinase AInhibitor 6-22 Amide were used as the inhibitors for the p60c-src kinaseand PKA kinase, respectively (Calbiochem, CA).

Results for the 194-target pool are shown in FIG. 5. The p60c-src andPKA substrates were phosphorylated selectively by p60c-src and PKAkinases; p60c-src and PKA were inhibited selectively by theircorresponding inhibitors. The p60c-src and PKA substrates were mixedwith 48 targets containing a tyrosine residue, 48 targets containing aserine residue, and 96 targets containing synthetically phosphorylatedtyrosine or serine residues. All the 96 non-phosphorylated targetsshowed signals equivalent to background, and all the 96 phosphorylatedtargets showed signals comparable to p60c-src and PKA substrates.Similar data was also obtained using pools of 6 or 100 targets.

FIG. 6 summarizes results for the 194-target pool. Cross reactivity forp60c-src and PKA was less than 1%. Furthermore, selective inhibition ofenzymatic activity of each kinase was observed. Specifically, Src KinaseInhibitor I showed 85% inhibition of p60c-src kinase while having nodetectable effect on PKA kinase and Protein Kinase A Inhibitor 6-22Amide showed greater than 99% inhibition of PKA while having nodetectable effect on p60c-src kinase.

To demonstrate the robustness of the universal detection method, aseries of control targets were made containing amino acid residues thatcould potentially interfere with the labeling method. The controltargets included substrates for other kinases, such as PKC, CAMKII, Abl,ERK, that are not typically phosphorylated by p60c-src and PKA kinases.As shown in FIG. 7, substrates for p60c-src kinase (circled in FIG. 7A)and substrates for PKA kinase (circled in FIG. 7B) showed high signal.The low background signal observed for the control targets in FIG. 7indicates that the chemical labeling method is specific for thephosphomonoester moiety.

To confirm that free carboxylic acid moieties did not interfere with thechemical labeling method, R5 (having an aspartic acid residue) and R6(having a glutamic acid residue) were mixed with the 96-target pool andthe four step assay repeated. The low pH, EDC pre-treatment was usedbefore the phosphomonoester labeling. Decreased R5 and R6 signals wereobserved while signal obtained for other targets were similar to thedata shown in FIG. 7.

To further demonstrate the reliability of the chemical labeling method,the anti-phosphotyrosine antibody was used side by side with theuniversal detection method. FIG. 8A shows the results of antibodylabeling, and FIG. 8B shows the results of the chemical labeling method.100% correlation was obtained using pair wise signal comparisons fortargets yielding positive phosphorylation intensities in chemical andantibody labeling methods. The results demonstrate that both detectionmethods correlated well.

Pattern Recognition as a Tool for Evaluating Kinase Activity

Data sets obtained from multiplex kinase assays can be quite complex,and in some cases, difficult to evaluate using a standard bar graph. Thedifficulty can be acerbated when comparing the phosphorylation state ofa panel of multiple targets resulting, from different treatments such asexposure to different kinases and/or kinase inhibitors. In such cases,it is helpful to represent the data in a format that is readilydistinguishable by pattern recognition. A representation that isconvenient for pattern recognition by eye is a “star plot.” A star plotis comparable to a bar graph except that the bars occur in a radialpattern, about a circular axis, rather than along a linear axis. Signalfrom each target appears as a spoke and the quantitative amount ofsignal is correlated to the length of the spoke.

FIG. 9 shows exemplary star plots for several kinases individuallyevaluated for the ability to phosphorylate targets in the same mixtureof 96 different protein-DNA targets. For each star plot, the 96 targetsare plotted in the same order and starting with the same target at the12 O'clock position. Absence of a spoke indicates that no signal wasdetected for the particular target. Where a spoke is present, its lengthindicates the intensity of the signal, which is proportional to thelevel of phosphorylation of the target. As shown in FIG. 9, the patternof activity is unique for each kinase and can be readily determined byeye. Comparison of the star plot for each kinase indicates that thetarget mixture contains specific protein substrates for each kinaseenzyme.

FIG. 10 shows an example of the data obtained by testing two kinases(p60c-src and PKA) with the target mixture containing 96 differentprotein-DNA targets and in the presence or absence of different kinaseinhibitors. Plots 1 and 2 of FIG. 10 show the signals obtained forphosphorylation of the target mixture with p60c-src and PKA,respectively. Plot 3 of FIG. 10 combines the data from plots 1 and 2,demonstrating the ease with which the individual kinase patterns can berecognized when combined. As shown in plots 4 and 5 of the figure,selective inhibition of enzymatic activity of each kinase was observed.As shown in plots 6 and 7 of FIG. 10, inclusion of an inhibitortargeting either p60c-src or PKA, in the dual-enzyme mixture, removedsignal from the expected target enzyme and restored the star plots tothe same pattern as the non-targeted enzyme.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

1-27. (canceled)
 28. A method of modifying a target compound, comprising(a) providing a target compound comprising a carboxylic acid moiety anda phosphomonoester moiety; (b) reacting the carboxylic acid moiety ofthe target compound with a first carbodiimide compound to form anN-acylurea moiety on the target compound, thereby forming acarboxyl-protected target compound, wherein the carboxyl-protectedtarget compound comprises the N-acyl urea moiety and thephosphomonoester moiety; and (c) reacting the phosphomonoester moiety ofthe carboxyl-protected target compound with a second carbodiimidecompound and an amine compound to form a phosphoric amide moiety on thecarboxyl-protected target compound.
 29. The method of claim 28, furthercomprising a step of (d) detecting the phosphoric amide moiety.
 30. Themethod of claim 28, wherein the amine compound comprises a label moiety.31. The method of claim 30, wherein the phosphoric amide moietycomprises the label moiety.
 32. The method of claim 31, furthercomprising a step of (d) detecting the label moiety of the phosphoricamide moiety.
 33. The method of claim 28, wherein the target compoundcomprises a protein.
 34. The method of claim 33, further comprisingidentifying the type of amino acid that is attached to the phosphoricamide moiety.
 35. The method of claim 33, further comprising identifyingthe primary sequence location of an amino acid that is attached to thephosphoric amide moiety.
 36. The method of claim 33, wherein theN-acylurea moiety comprises the alpha carboxylic acid moiety of theC-terminal amino acid of the protein, the beta carboxylic acid moiety ofaspartate or the gamma carboxylic acid moiety of glutamate.
 37. Themethod of claim 33, wherein the phosphoric amide moiety is selected fromthe group consisting of phosphoserine, phosphothreonine, andphosphotyrosine.
 38. The method of claim 33, wherein step (a) furthercomprises contacting the protein with a kinase under conditions forproducing the phosphomonoester moiety of the protein.
 39. The method ofclaim 38, further comprising identifying a phosphorylation activity ofthe kinase from the phosphoric amide moiety of the carboxyl-protectedtarget compound.
 40. The method of claim 38, wherein the protein iscontacted with the kinase in the presence of a kinase inhibitor or akinase activator.
 41. The method of claim 40, further comprisingidentifying inhibition or activation of phosphorylation activity of thekinase from the phosphoric amide moiety of the carboxyl-protected targetcompound.
 42. The method of claim 28, wherein the carbodiimide compoundis EDC.
 43. The method of claim 28, wherein the amine compound isattached to a solid support.
 44. The method of claim 28, wherein thefirst carbodiimide compound and the second carbodiimide compoundcomprise the same molecular species.
 45. The method of claim 28, whereinthe first carbodiimide compound is contacted with the target compound ina reaction vessel and the second carbodiimide compound is contacted withthe carboxyl-protected target compound in the reaction vessel.
 46. Themethod of claim 45, wherein the first carbodiimide compound and thesecond carbodiimide compound are simultaneously added to the reactionvessel.
 47. The method of claim 28, wherein the phosphoric amide moietyis attached to a solid support.